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Previous Article | Next Article 
Molecular and Cellular Biology, September 2003, p. 6507-6519, Vol. 23, No. 18
0270-7306/03/$08.00+0 DOI: 10.1128/MCB.23.18.6507-6519.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.
Bin He,1 and Georg F. Weber1,2*
Department of Radiation Oncology, New England Medical Center,1 Sackler School of Graduate Biomedical Sciences, Tufts University School of Medicine, Boston, Massachusetts2
Received 10 December 2002/ Returned for modification 20 February 2003/ Accepted 19 June 2003
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ABSTRACT |
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INTRODUCTION |
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Gain-of-function mutations in the epidermal growth factor (EGF) family of receptors and their associated pathways of signal transduction often underlie the transformation of breast tissue, as is evidenced by the cases of breast cancers that overexpress the EGF family receptor Her-2/neu. This dysregulation is also prominent in steroid hormone-independent breast cancer, where excessive activation of EGF receptor pathways may be the only driving force for cell cycle progression (5). The intracellular signal transduction associated with members of the EGF receptor family is mediated by multiple proto-oncogene products, including protein kinase C, phosphatidylinositol 3-kinase (PI 3-kinase), and Akt kinase (31, 35). Their constitutive activation occurs as a consequence of overexpression of Her-2/neu (6, 24, 33, 41, 56) and may be sufficient to cause transformation.
Expression of the cytokine osteopontin is necessary and may be sufficient for the formation of metastases by breast cancer. High levels of osteopontin in the disease are an adverse prognostic factor (42, 45). Multiple metastatic breast cancer cell lines express osteopontin, and transfection of the osteopontin gene into weakly tumorigenic human breast tumor cell lines confers invasive behavior (47, 50, 51). Increasing the expression of osteopontin or transfection of osteopontin-encoding cDNA into a previously benign cell line is sufficient to produce a metastatic phenotype in a rat mammary model (38). Short regulatory DNAs exist in human cancer cells that can be transferred into model rat mammary cell lines and can induce metastatic spread. These noncoding fragments of DNA act via the common effector gene osteopontin (4, 11, 19, 20).
Receptor ligation by EGF can induce osteopontin gene expression (2, 34) through signal transduction that proceeds via protein kinase C and tyrosine kinases (8). This implies that gain-of-function mutations in the EGF receptor pathway in breast cancer, causing dysregulated growth, may also mediate the overexpression of osteopontin, leading to dissemination. We find osteopontin to be constitutively expressed in malignant but not in benign transformed breast cells. Here, we trace the cause for this to constitutive activation of Akt kinase, an enzyme that is part of the EGF signaling pathway.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Induction of growth factor signaling. Cells were plated at 5 x 105 per well in 6-well plates. After 7 h, cultures were washed twice with phosphate-buffered saline and maintained in growth factor-deprived medium (containing 0.05% bovine serum albumin) for 14 h. The cells were then stimulated with the indicated amounts of EGF (obtained from Sigma, St. Louis, Mo.) for the indicated time frames before harvesting and analysis.
To assess candidate signal transduction molecules involved in the induction of osteopontin gene expression, specific inhibitors were added to the cells. The PI 3-kinase inhibitor wortmannin and the phospholipase C (PLC) inhibitor U73122 were purchased from Calbiochem (San Diego, Calif.). The cells were pretreated with the respective inhibitors or vehicle (dimethyl sulfoxide) alone for 30 min and then cotreated with EGF for an additional 8 h. Preliminary dose-response experiments had established that the concentrations of 100 nM wortmannin and 500 nM U73122 were effective and nontoxic.
For the investigation of cell growth rates, each cell line was plated at 5,000 cells/well in 24-well plates. Daily, five wells per group were harvested by trypsinization, and the cell numbers were determined in a Coulter Z-Series Counter. At each time point, the cell numbers from the five wells of the various groups of transfectants were analyzed for statistically significant differences by the Wilcoxon-Mann-Whitney test, by accepting a probability of error of <5%. The cell viability was always higher than 95%.
RNase protection assay.
Osteopontin expression was analyzed by an RNase protection assay on total RNA by using a 400-bp probe from the 3' region of the transcript and controlling for loading with a probe for ß-actin. Total RNA was isolated using the RNeasy Mini Kit (QIAGEN GmbH, Hilden, Germany), and its amount and purity were confirmed by spectrophotometry. The RNase protection assay was performed with a commercial kit (Ambion). Briefly, [
-32P]UTP-labeled probes were mixed with sample RNA and coprecipitated. Hybridization proceeded for 10 min at 68°C, followed by digestion of the unprotected RNA with RNase A-T1 for 30 min at 37°C and separation of the hybridized RNA on 5% acrylamide-8 M urea gels. The gels were dried on filter paper in a gel dryer (Bio-Rad) and exposed to X-ray film. Autoradiographs were scanned using an AlphaImager 2200 spot densitometer (Alpha Innotech Corporation), and the integrated densities of areas were recorded.
Northern blotting. Total RNA was isolated by using the RNeasy Mini Kit (QIAGEN GmbH) according to the manufacturer's instructions. Purity and yield were confirmed by spectrophotometry at 260 and 280 nm. Ten micrograms of RNA was denatured and separated on formaldehyde-containing 1.2% agarose gels by electrophoresis, blotted onto nylon membranes (Amersham), and probed with a 32P-labeled osteopontin cDNA probe (generated by PCR using forward primer 5'-GCGAATTCTGGCTTATGGACTGAGGTC-3' and reverse primer 5'-ATGGATCCGGAACTGTGTTTTTGCCTC-3') or a 36B4 cDNA probe (generously provided by Goberdhan Dimri). Hybridization of the probe to the RNA was allowed to proceed overnight, and the membranes were washed and exposed to X-ray film at -80°C before development.
Immunoblot assay. For analysis of secreted osteopontin, the growth factor-deprived cell culture supernatant was collected from each treatment group. Forty microliters of supernatant per sample was electrophoresed on sodium dodecyl sulfate (SDS)-10% polyacrylamide minigels with nonreducing sample buffer. For analysis of intracellular proteins, cells were lysed in radioimmunoprecipitation assay buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) for the osteopontin blot or Akt kinase blot or in NP-40 buffer (150 mM NaCl, 1 mM EDTA, 20 mM Tris-HCl [pH 8.0], 0.5% NP-40) for the phospho-Akt blot. Cell lysates at equal amounts of protein (10 to 20 µg/lane) were electrophoresed on reducing SDS-10% polyacrylamide gels. The separated proteins were transferred to Pure Nitrocellulose membranes (Osmonics Inc.) and probed with antibodies to osteopontin (R&D Systems), Akt, and Akt phosphorylated at Ser473 (New England Biolabs, Beverly, Mass.). Detection was accomplished with the ECL chemiluminescence reagent.
Reporter assays. Comma-D cells stably transfected with vector, wild-type Akt, or constitutively active Akt were plated at 5 x 105 cells per 60-mm-diameter petri dish and grown for 24 h before transfection with Fugene. Various reporter constructs containing the luciferase reporter gene under the control of truncation mutants of the osteopontin promoter were used at 2 µg per transfection. The common internal transfection standard pRL-SV40 (10 ng per transfection) served as a control for transfection efficiency. Twenty-four hours after transfection, the cells were harvested in 0.4 ml of reporter lysis buffer (Promega), and dual luciferase reporter assays were performed by following the protocol provided by the manufacturer. Forty microliters of lysate was used for measurement in a luminometer (Turner Designs TD-20/20). Three separate experiments were performed, and results were calculated as means ± standard deviations. The lengths of the osteopontin promoter fragments tested were -88 to +79, -258 to +79, -472 to +79, -600 to +79, -777 to +79, and -882 to +79 (23).
DNA constructs and infection. The constructs of Akt kinase, constitutively active Akt kinase, and dominant-negative Akt kinase (K179M) in the pCMV-6 vector or in the retrovirus vector pLNCX were generously provided by Thomas Franke (12, 32) (the constitutively active Akt kinase was recloned into pLNCX in our laboratory). The construct for expression of the mouse osteopontin coding sequence was obtained by reverse transcription-PCR from osteosarcoma cells by using the sense primer 5'-ATGGATCCCGACCATGAGATTGGCAG-3' and the antisense primer 5'-GCGAATTCGGACTGTGTTTTTGCCTC-3'. The resultant BamHI-EcoRI cDNA fragment was ligated into the retrovirus vector pBabe. The construct for expression of osteopontin antisense, a full-length murine osteopontin cDNA generated by PCR, was ligated in the antisense orientation into the EcoRI and BamHI sites of pBabe. The integrity of all constructs was confirmed by DNA sequencing.
The packaging cell line tsa54 was used to generate high-titer retrovirus (16) for infection of all breast epithelial cell lines. Ten micrograms of each retrovirus construct in conjunction with 10 µg of the pIK packaging vector was transfected into tsa54 cells by the calcium phosphate coprecipitation method, and the culture supernatant was collected on the day following transfection. In order to generate stable Akt transfectants, the murine breast tumor cell lines were infected with virus-containing supernatants in the presence of 8 µg of Polybrene (Sigma)/ml. Infected cells were selected in MECL medium with G418 (200 to 500 µg/ml). For double infection, cells expressing constitutively active Akt or mutant Akt were infected with plasmids encoding sense or antisense osteopontin. Forty-eight hours later, selection with puromycin (2 to 4 µg/ml) commenced for 5 days, followed by analysis of RNA and protein expression. Several clones were selected and characterized for all single infectants, while the doubly transduced cells were selected and analyzed in bulk.
Cell motility. The cell motility experiment is often referred to as an in vitro wounding assay (21, 27) because a cell monolayer is disrupted and the rate of repopulation is assessed. Breast epithelial cells were plated at 2.5 x 105 per 100-mm-diameter petri dish. After 2 days, they were grown in growth factor-deprived medium for 24 h. The cultured cells were disrupted by scratching with a pipet aid equipped with a 1,000-µl tip. Repopulation of the cell-free area was examined under an inverted microscope after 22 h. In this time frame, cell division is negligible in growth factor-deprived medium.
Soft-agar growth. Cells (5 x 105 per 60-mm-diameter dish) were plated in triplicate with a top layer of 0.3% agar (Bacto Agar; Difco, Detroit, Mich.) and a bottom layer of 0.5% agar (both in MECL medium). Every other day, 0.3 ml of MECL medium was supplemented and the plates were examined microscopically for growth. After 3 weeks, the number of cell clusters per microscopic field and the number of cells per cluster were counted in five views per plate (one central, four peripheral), and cell clusters were photographed. Soft-agar assay conditions have not been standardized because they depend on the specific cell lines under study. The size of the cell clusters obtained differs widely with the cell lines used; cell clusters may comprise fewer than 10 cells (7, 25) or reach microscopically (36) or macroscopically (43) visible diameters. For Comma-D cells transfected with Akt, an incubation time of 3 weeks before enumeration has been found suitable (40). We chose to determine the frequency of clones formed and the clone size (numbers of cells per cluster). At the plating density and within the time frame employed here, this quantitation could be performed accurately, and the resulting histograms of cluster size and frequency were considered to be more informative than the simple enumeration of clusters above a randomly chosen size. The resulting histograms are characteristic of the individual cell lines and were analyzed for statistical differences in variance by the F-test, by accepting a probability of error of 1, 5, or 10% with a one-sided paired comparison.
In vivo tumorigenesis. MT2994 cells or FSK-7 cells were harvested by trypsinization. A total of 0.5 x 106 cells in 0.5 ml of phosphate-buffered saline were injected orthotopically into the left inguinal mammary fat pads of female BALB/c mice (age, 8 to 10 weeks). After 21 days, the animals were sacrificed and necropsy was performed for the detection of primary tumors or hyperplasia and metastases. To ascertain that the FSK-7 cells were free of murine pathogens, serum samples from injected animals were tested for Sendai virus, pneumonia virus of mice, mouse hepatitis virus, minute virus of mice, Theiler's murine encephalomyelitis virus, reovirus, Mycoplasma pulmonis, mouse parvovirus, epizootic diarrhea of infant mice virus, lymphocytic choriomeningitis virus, mouse adenovirus, ectromelia (mousepox virus), K virus, polyomavirus, mouse thymic virus, mouse cytomegalovirus, hantavirus, Encephalitozoon cuniculi, and cilium-associated respiratory bacillus. Results of serological testing were negative in all instances. The in vivo experiments were performed under a protocol approved by the institutional animal care and use committee at Tufts-New England Medical Center.
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RESULTS |
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The constitutive osteopontin expression in malignant breast epithelial cells correlates with a gain of function in the PI 3-kinase pathway. We analyzed the involvement of signal transduction molecules associated with the EGF receptor in the induction of osteopontin gene expression. Addition of the PLC inhibitor U73122 or the PI 3-kinase inhibitor wortmannin reversed the EGF-mediated increase in osteopontin mRNA levels in Comma-D and FSK-7 cells. In contrast, the high abundance of osteopontin mRNA in MT2994 cells remained unaffected by either inhibitor (Fig. 3A).
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Overexpression of Akt kinase induces osteopontin gene expression. To gain further mechanistic insight into the contributions by Akt kinase to osteopontin gene expression, we stably transduced the breast epithelial cells with various Akt kinase constructs. Comma-D cells were transfected with vector, wild-type Akt, or constitutively active Akt. FSK-7 cells were transfected with vector or constitutively active Akt. MT2994 cells were transfected with vector, wild-type Akt, or a dominant-negative Akt mutant. The expression levels of the transfected genes were measured on the RNA and protein levels. As expected, osteopontin was constitutively expressed in Akt kinase-transduced Comma-D cells and FSK-7 cells, while the constitutive osteopontin gene expression in MT2994 cells was suppressed by the dominant-negative Akt kinase mutant, as shown by an RNase protection assay and Western blotting (Fig. 4A). We confirmed the osteopontin message levels by Northern blotting (with 36B4 as a loading control) and found this measurement to be in good correlation with the RNase protection assay results (Fig. 4B). The levels of osteopontin induced by wild-type Akt and constitutively active Akt are comparable, likely reflecting the substantial overexpression of Akt kinase. At these amounts, the baseline activity of wild-type Akt kinase is sufficient to transduce a signal. It should be noted that the reduction of osteopontin gene expression by dominant-negative Akt kinase is partial. Only moderate levels of overexpression can be achieved for dominant-negative Akt kinase, because this mutant also slows down cell division (compare Fig. 5B below), consistent with the hypothesis that the constitutive activation of Akt kinase in MT2994 cells is causative for the transformation of these cells.
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Akt kinase enhances the growth rate through osteopontin-independent pathways and mediates migration through osteopontin. In order to differentiate between osteopontin-dependent and osteopontin-independent consequences of Akt kinase overexpression, we generated doubly transduced lines. FSK-7 cells that stably expressed constitutively active Akt kinase were infected with a retrovirus containing osteopontin antisense, yielding FSK-7 vector plus vector, FSK-7 constitutively active Akt plus vector, and FSK-7 constitutively active Akt plus antisense osteopontin cells. Similarly, we generated MT2994 vector plus vector, MT2994 dominant-negative Akt plus vector, and MT2994 dominant-negative Akt plus osteopontin cells. As before, the expression levels of the modulated genes were measured by Western blotting. The elevated osteopontin expression in transfectants of constitutively active Akt kinase could be reversed by cotransfection of antisense osteopontin, and the suppression of osteopontin by dominant-negative Akt was reversed by cotransfection with the osteopontin gene (Fig. 5A).
We tested the growth rates of doubly transduced cells by plating 5,000 cells per well in 24-well plates, followed by daily cell counts in quintuplicate. Remarkably, within only 1 day, transfection of constitutively active Akt kinase into FSK-7 cells significantly induced the growth rate, while transfection of dominant-negative Akt kinase into MT2994 cells significantly reduced it. The differences remained significant throughout the 4 days of measurement. In contrast, the stable transfection of osteopontin into cells overexpressing dominant-negative Akt kinase or of osteopontin antisense into cells overexpressing constitutively active Akt kinase had no effect on the growth rates (Fig. 5B). Identical results were obtained with singly transduced FSK-7 cells expressing constitutively active Akt kinase and MT2994 cells expressing dominant-negative Akt kinase (data not shown).
We also analyzed cell motility in vitro. Doubly transduced FSK-7 cells were plated at 2.5 x 105 per 100-mm-diameter dish and grown for 2 days. They were starved for 24 h in growth factor-deprived medium. The geometry of the cell culture was disturbed by scratching across the diameter of the cell culture dish. After 22 h, FSK-7 cells transfected with constitutively active Akt displayed reorientation and migration into the space between the continuous layers. Cotransfected osteopontin antisense completely reversed this effect, and like cells transfected with the vector control, these cells did not move (Fig. 5C). For a more quantitative analysis of cell motility, we performed a transwell chamber chemokinesis assay with coating of both sides of the membrane with 10 µg of fibronectin/ml. A total of 2.5 x 104 cells were placed in the upper chamber, and serum- and growth factor-free medium was placed in both compartments. After 4 h, the numbers of cells per optical field on the lower side of the membrane were 9 ± 2 for FSK-7 vector plus vector, 19 ± 6 for FSK-7 vector plus antisense osteopontin, 51 ± 2 for FSK-7 constitutively active Akt plus vector, and 32 ± 4 for FSK-7 constitutively active Akt plus antisense osteopontin. These results are consistent with osteopontin-independent roles of Akt as a growth-promoting gene product and osteopontin-dependent roles of Akt as a migration-promoting gene product.
Osteopontin-dependent and -independent pathways contribute to generating a malignant phenotype. Because osteopontin is necessary for soft-agar colony formation (see Fig. 1C) and Akt kinase induces osteopontin expression, we asked whether the constitutive overexpression of Akt kinase in FSK-7 cells was sufficient to confer anchorage-independent growth. As expected, Akt-transfected FSK-7 cells displayed clone formation in soft agar, whereas vector-transfected FSK-7 cells did not. The repeated addition of an anti-osteopontin antibody to the cell culture reduced the numbers and sizes of clones formed. In contrast, a control immunoglobulin had no effect (Fig. 6A). As in the preceding experiment (compare Fig. 1C), the inhibition of clone formation by an antiosteopontin antibody was partial, a finding which may reflect limitations in the neutralization by the antibody. We therefore analyzed the doubly transduced cells for colony formation in soft agar. Coexpression of osteopontin antisense in FSK-7 cells expressing constitutively active Akt led to a partial reduction in colony formation. Consistent with the earlier results, FSK-7 cells transduced with vector constructs did not form clones in soft agar (Fig. 6B). In a complementary approach, expression of dominant-negative Akt in MT2994 cells caused a partial reduction in the numbers and sizes of clones formed (a shift of the histogram to the left), consistent with the partial suppression of Akt kinase activity by the dominant-negative construct. Coexpression of osteopontin virtually completely reversed this inhibition (Fig. 6C). The differences among the transduced MT2994 cells did not reach statistical significance, which is likely due to the incomplete suppression of endogenous Akt activity by the dominant-negative construct. Dominant-negative variants of essential growth-promoting gene products can typically be overexpressed only at moderate levels (1). Residual Akt activity has previously been observed after transfection with the Akt (K179M) construct (12, 32).
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Metastasis formation arises with consistent patterns of organ preference. It has not been clear how the primary defect of growth dysregulation is linked to the expression of specific metastasis genes. We have screened signal transduction molecules associated with the EGF receptor and have identified constitutive phosphorylation, indicative of activation, of Akt kinase in malignant, but not in benign growth-dysregulated, immortal breast epithelial cells. Akt kinase activity supports tumor growth as well as the induction of osteopontin, a metastasis gene that is essential for breast cancer dissemination to bone. Our results may provide a molecular explanation for the previous observation that transfection of Akt1 into immortalized, not fully transformed Comma-1D cells mediates, mutually independently, the transformation and invasion of breast epithelial cells (40). Human breast carcinogenesis is associated with a loss of cellular retinal-binding protein (CRBP) expression in about one-fourth of all cases. The inhibition of the PI 3-kinase/Akt kinase pathway by CRBP suppresses breast cancer cell survival and anchorage-independent growth (30). The latter is likely due to the inhibition of osteopontin gene expression. Elevated Akt kinase activity is also seen in metastasizing, but not in nonmetastasizing, liver tumor cells (36). Akt kinase has also been associated with the invasiveness of pancreas cancer, but here the pro-metastatic gene induced by Akt is the IGF-I receptor (48).
The cause for the constitutive activation of Akt kinase in MT2994 cells remains unknown. Activating mutations in Akt itself seem unlikely, because mutants with known activating amino acid changes, such as E40K, still respond to inhibition by wortmanin. The loss of negative regulators of Akt kinase may desensitize the enzyme and generate the appearance of PI 3-kinase independence. We have sequenced the mRNA of PTEN, the most prominent negative regulator of Akt, in MT2994 cells and FSK-7 cells (data not shown). No mutations were found in either case. Reduced expression levels of PTEN or a lack of other negative regulators are likely causes.
Our studies of murine transformed breast epithelial cells have indicated that constitutive activation of Akt kinase, independent of PI 3-kinase, causes a malignant phenotype through continuing cell cycle progression and expression of osteopontin (MT2994 cells). In contrast, breast epithelial cells may be immortalized through signal transduction that is independent of the EGF/PLC/PI 3-kinase/Akt kinase pathway (Comma-D cells and FSK-7 cells), but this transformation is not associated with osteopontin gene expression or with metastasis. Dysregulation of Akt kinase activity also plays a prominent role in human breast tumor cells. There, the constitutive activation of Akt kinase is typically secondary to overexpression of ErbB on the cell surface and is dependent on PI 3-kinase. Estrogen stimulates Akt activation, which is reflected in phosphorylation at serine 473 of the oncoprotein, in estrogen receptor-negative breast cancer cells. Activation of Akt by estrogen in these cells is time and dose dependent and can be blocked by inhibitors of PI 3-kinase and Src kinase but not by estrogen antagonists (49). The interdependent regulation between the estrogen receptor and PI 3-kinase/Akt pathways may play an important role in human breast carcinogenesis and could contribute to ligand-independent breast cancer cell growth (46). Here we describe a single molecular defect responsible for both growth dysregulation and induction of metastatic potential in breast cancer cells. It enhances our molecular insights into carcinogenesis and defines candidate targets for therapeutic intervention.
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ACKNOWLEDGMENTS |
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We are grateful to Joseph Jerry for kindly providing the cell lines. We thank Thomas Franke for generously sharing the Akt kinase constructs and David Denhardt for supplying the osteopontin reporter constructs.
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FOOTNOTES |
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Present address: Department of Gastroenterology, First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, People's Republic of China.
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